AlanFung:LabNotes/Probe Set Quantification/2009-6-3: Difference between revisions
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[[Image:High Range OliGreen Standard.jpg]] | [[Image:High Range OliGreen Standard.jpg]] | ||
==Step 4: Prepare OliGreen Reagent== | ==Step 4: Prepare OliGreen Reagent== | ||
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*Subtract the fluorescence value of the reagent blank from that of each of the samples | *Subtract the fluorescence value of the reagent blank from that of each of the samples | ||
*Determine the oligonucleotide concentration of the sample from the standard curve generated in Oligonucleotide Standard Curve | *Determine the oligonucleotide concentration of the sample from the standard curve generated in Oligonucleotide Standard Curve | ||
==Data== | |||
[[Image:OliGreen Test 06_03_09.jpg]] | |||
==Discussion== | |||
The data looks quite reasonable, but still some of the data points doesn't make sense | |||
==Improvements== | |||
*Perform 3 replicate for each concentration | |||
*Add in the OliGreen dye in the dark and prepare the reaction as quick as possible |
Latest revision as of 18:11, 3 June 2009
Probe Preparation[edit]
Objective[edit]
- Perform only the high-range standard curve
Samples & Materials[edit]
- Quant-iT OliGreen ss DNA Assay Kit (Cat. no. O11492)
*Quant-iT OliGreen ss DNA Reagent (Component A), solution indimethylsulfoxide (DMSO) *20X TE Buffer (Component B) *Oligonucleotide Standard (Component C) 18-base M13 sequence primer, with the sequence 5'-TGTAAAACGACGGCCAGT-3'
- Sterile, distilled, DNase-free water
Overview[edit]
Procedures[edit]
Step 1: Preparing Working Reagent[edit]
*First Time
- Allow the reagent to warm to RT before opening the vial
- Aliquot reagent dye to 0.5mLtubes (2.2uL each tube) - Stor at -20C
*First Time
- Dilute 20X TE buffer to 1X working solution
*Take 1mL 20X TE buffer and dilute it with 19mL DNase-free water
- Determine the volume of Working Reagent Required
(# standards (5) + # samples) x (replicates (2)) x (volume per well (100 μL)) One tube of aliquot gives you 440uL of OliGreen dye
Step 2: Prepare Oligonucleotide Standard[edit]
- Dilute the oligonucleotide standard provided(100ug/mL) to 100ng/mL with 1XTE buffer
File:High Range OliGreen Standard.jpg
Step 4: Prepare OliGreen Reagent[edit]
- Prepare an aqueous working solution of the Quant-iT OliGreen reagent by making a 200 fold dilution of the concentrated DMSO solution in 1XTE Buffer
- Add 437.8uL of 1X TE Buffer to the 2.2uL dye aliquot
Step 5: Sample Anaylysis[edit]
- Vortex all samples and reagent really well before loading
- Add 25uL of the aqueous working solution of the Quant-T OliGreen reagent to each sample and standard
- Load both samples and standard and into strip tubes
- The strip tubes should look like this:
- with the letters A-E (in A1-B2) being the standards and the numbers 1-11 (in C2-H4) being the samples
- (it is read top-to-bottom then left-to-right; A1 – H1, A2 - H2, etc)
- Mix well and incubate for 5 mins at RT, protected from light
- After incubation, measure the sample fluorescence using the RT-PCR
- Standard fluorescein wavelength (excitation ~480nm, emmision ~520nm)
- Measure the absorbance at 520nm on the RT-PCR
- For the Chromo4 Real Time PCR
- Open the Opticon Monitor 3 program
- Perform the following
Incubate at 25C 4M 50S Incubate at 25C 10S Plate Read Go back to line 2 for one more time END
- Subtract the fluorescnece value of the reagent blank from that of each of the samples
- Use corrected data to generate a standard curve of fluorescence versus oligonucleotide concentration.
- Record the ssDNA concentrations calculated by the worksheet
- Incubate for 5 mins at room temperature, protected from light
- Measure the fluorescence of the sample
- Subtract the fluorescence value of the reagent blank from that of each of the samples
- Determine the oligonucleotide concentration of the sample from the standard curve generated in Oligonucleotide Standard Curve
Data[edit]
File:OliGreen Test 06 03 09.jpg
Discussion[edit]
The data looks quite reasonable, but still some of the data points doesn't make sense
Improvements[edit]
- Perform 3 replicate for each concentration
- Add in the OliGreen dye in the dark and prepare the reaction as quick as possible